Control and Regulation of KplE1 Prophage Site-specific Recombination

Gaël Panis,Mireille Ansaldi,Vincent Méjean

doi:10.1074/jbc.m701827200

Abstract

KplE1 is one of the 10 prophage regions of Escherichia coli K12, located at 2464 kb on the chromosome. KplE1 is defective for lysis, but it is fully competent for excisive recombination. In this study, we have mapped the binding sites of the recombination proteins, namely IntS, TorI, and IHF on attL and attR, and the organization of these sites suggests that the intasome is architecturally different from the lambda canonical form. We also measured the relative contribution of these proteins to both excisive and integrative recombination by using a quantitative in vitro assay. These experiments show a requirement of the TorI excisionase for excisive recombination and of the IntS integrase for both integration and excision. Moreover, we observed a strong influence of the supercoiled state of the substrates. The KplE1 recombination module, composed of the integrase and excisionase genes together with the attL and attR DNA regions, is highly similar to that of several phages infecting various E. coli strains as well as Shigella flexneri and Shigella sonnei. The in vitro recombination data reveal that HK620 and KplE1 att sequences are exchangeable. This study thus defines a new site-specific recombination module, and implications for the mechanism and regulation of recombination are discussed.

Highlights

Phage ␭ has long served as a model system for studies of regulated site-specific recombination (1)
We present a detailed analysis of the recombination sites of KplE1 that are highly homologous to that of Sf6 and HK620 phages and of strains K1 RS218, UTI89, APEC-O1, and SsO46 prophages
The KplE1 prophage of E. coli K12 is a defective prophage that cannot form infectious particles, yet it is competent for genome excision (27)

Summary

A NEW RECOMBINATION MODULE ANALYZED*

The KplE1 recombination module, composed of the integrase and excisionase genes together with the attL and attR DNA regions, is highly similar to that of several phages infecting various E. coli strains as well as Shigella flexneri and Shigella sonnei. In response to a change in the physiological state of the bacteria, mainly in response to stress conditions such as DNA damage, ␭ phage DNA is excised from the host chromosome This excisive reaction recombines attL and attR to restore the attP and attB sites on the circular ␭ and Escherichia coli. Prophages occasionally mutate to loose some of the functions essential for lytic growth, in which case the strain is no longer able to liberate infectious particles (20) Such prophages are termed defective, but they can inactivate host genes or conserve genes important for host pathogenicity or fitness. The boundaries of KplE1 prophage are 16-bp-duplicated core sequences (CTGCAGGGGACACCAT) separated by 10.216 kb outlined by the argW tRNA gene and the dsdC gene, and com-

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

47 This work Novagen

DISCUSSION